The present invention relates to apparatus for performing the incubation and washing requirements of solid-phase, binder-substrate analysis of clinical substances.
A larger number of different types of tests are performed on various clinical substances such as human serum by using binder-substrate analysis. In such an analysis, a binding agent is typically suspended in a suppportive medium to which might be added either a quantitated standard substrate or the unquantitated substrate in the clinical substance. Additionally, a measure of labeled substrate, as nearly identical to the substrate of the standard and clinical substance as possible, is added to the medium to serve as a marker for the distribution of all substrate during incubation. This supportive medium is then incubated under precisely controlled conditions favoring the equilibrium of binder-substrate complex and the individual components. After the incubation has achieved a point of equilibrium, some method of separation is performed allowing isolation of either the binder-substrate complex or the free components.
Since the labeled substrate is selected for those pertinent similarities to the standard and unquantitated substrate, it will distribute itself exactly like those unlabeled substrates at equilibrium. Measurement by suitable means of that label in the isolated fraction will provide the same results as if the total (labeled and unlabeled) substrate in the isolated fraction is measured. Comparison of the amount of substrate found in the isolate of the unquantitated clinical substances to that of the standards provides the result of the analysis.
One such method of separation, isolating the binder-substrate complex from the components, is termed "solid phase". Solid phase defines the physical condition of the binder-substrate complex at the point of equilibrium when the separation step is to occur. Typically, the binder is originally the solid phase component. When incubation is terminated, the separation step involves isolation of the solid phase binder-substrate complex from the liquid phase of the assay medium. If the binder is physically attached to microscopic-solid particles, the separation would involve a centrifuge. If the binder is attached to a large enough particle such as a 1/4 inch dia. sphere or the bottom interior surface of a test tube, the separation step involves decantation or aspiration of the liquid phase from the immobilized, solid-phase binder-substrate complex.
The application of solid phase to the binder-substrate analysis has eliminated the centrifuge in several cases (where large enough supports are used); but added to the procedure, time and/or temperature enhancement of the incubation step, and a wash step(s) to adequately isolate the solid phase complex from the liquid phase components. These additional requirements must be controlled to guarantee reproducibility between all specimens.
One such application of solid-phase binder-substrate analysis determines the presence of hepatitis virus in serum specimen. The primary binder is an antibody to hepatitis virus, attached to a 1/4 inch dia. bead. This solid-phase binder is immersed in a buffered medium containing a measure of specimen, all contained within a test tube.
The test tube is maintained at a controlled temperature for sufficient time (incubation) to allow optimum formation of the antibody-virus complex on the surface of the plastic bead. The liquid medium containing all unreacted substances is then removed from the test tube and the bead and inner surfaces of the test tube are adequately washed leaving the solid phase antibody-virus complex. A second antibody, similar in binding properties to the primary but solubilized in a buffered medium, is then added to the test tube containing the solid phase complex. Additionally the second antibody is suitably labeled to allow subsequent detection. The test tube is again maintained according to those parameters optimizing the formation of a second binding; the virus by the labeled antibody. The only virus available to this second, labeled antibody is carried over through the washing(s) from the first incubation. Therefore, any secondary binding will join the labeled antibody to the solid-phase antibody-virus complex forming a sandwich. The liquid medium containing all unreacted substances is again removed from the test tube and the bead and inner surfaces of the test tube are washed leaving the solid-phase primary antibody-virus-labeled antibody complex. The plastic bead is then transfered to a carrier-tube suitable to the label detection apparatus. Detection of label on the plastic bead implies presence of virus since no other form of labeled antibody is carried over through the washing(s) from the second incubation.
At present, the incubations, washings and drying of the solid phase components in this variety of binder-substrate analysis are generally performed by hand. The test consumes a large amount of technician time, thus labor costs are relatively high. The large number of manual steps often cause errors in the testing procedures. Variations in the parameters of incubation time and temperature, and wash volumes, inherent in manual processing, produces nonuniformity in test results. A further problem presented by extensive manual manipulation is cross-contamination of the samples and the constant exposure of the technician to potential biological and radioactive hazards.